ELISA
ELISA is a specific and sensitive method for quantifying proteins and detecting particular antigens or antibodies in solutions [2, 3]. ELISA is typically performed in 96-well polystyrene plates and consists of the stepwise binding of capture antibodies, the target analyte and detection antibodies with washing steps to separate bound and free sample components and incubation in an enzyme reaction buffer (e.g. substrate reaction solution) to create a detectable product in the soluble phase [15]. The most commonly used reporter enzyme are horseradish peroxidase HRP and alkaline phosphatase (AP) [2, 3]. A large selection of substrates are available including chromogenic, fluorescent and chemiluminescent compounds [15]. Complete ELISA kits are also commercially available for detection of specific cytokines and other targets. Reporter enzyme such as AP and HRP can be covalently attached to streptavidin [16] that binds with a high affinity to biotin which can be covalently attached to the detection antibody using NHS coupling [17]. The detection limits for ELISAs are usually in the lower nanogram (ng) range.
RIA
In a related method termed radio immunometric assay (RIA), the capture antibody may be saturated with a radioactive analyte that is displaced by the sample analyte and the released radioactivity measured from the liquid phase [18]. At present the RIA is a most sensitive technology for detecting well characterized molecules but requires the use of radioisotopes that increase the administrative burden and cost of performing assays while limiting their broad application. RIA can reach sensitivities limited only by the background radioactivity and can commonly reach pico mol and even as low as femto mol levels.
LC-MS/MS
Many compounds may enter the gas phase by electrospray ionization [19]. Where pure compounds are available, the direct use of mass spectrometry may reach the atto mol levels [20, 21]. For complex biological samples, detection of the parent ions by SIM, or of the fragment ions by SRM, has been shown to provide sensitivity as low as ng/ml [22, 23]. However most of the protein assays by SRM are of relatively high abundance in the micrograms per ml (μg/ml) [24] well above the concentrations typically reached by ELISA with ECL or colorimetric detection and the SRM method frequently suffers a lack of sensitivity in complex samples [25]. A liquid chromatography column coupled to an electrospray source or nanospray source or MALDI source for a tandem mass spectrometer can directly measure ionized peptides or small molecules with a sensitivity of about 1 μg or less in the low femto mol to pico mol range depending on the molecule [26].
There remains a need to measure molecules such as proteins that are present in low concentrations below those detectable by enzyme linked Immuno absorbent assays (ELISA).